U.S. patent application number 11/323261 was filed with the patent office on 2007-10-25 for prevention and reduction of blood loss.
Invention is credited to Shirish Hirani, Robert C. Ladner, Arthur C. Ley, Anthony Williams.
Application Number | 20070249807 11/323261 |
Document ID | / |
Family ID | 38535763 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070249807 |
Kind Code |
A1 |
Ladner; Robert C. ; et
al. |
October 25, 2007 |
PREVENTION AND REDUCTION OF BLOOD LOSS
Abstract
Methods are described for preventing or reducing ischemia and/or
systemic inflammatory response in a patient such as perioperative
blood loss and/or systemic inflammatory response in a patient
subjected to cardiothoracic surgery, e.g. coronary artery bypass
grafting and other surgical procedures, especially when such
procedures involve extra-corporeal circulation, such as
cardiopulmonary bypass.
Inventors: |
Ladner; Robert C.;
(Ijamsville, MD) ; Ley; Arthur C.; (Newton,
MA) ; Hirani; Shirish; (Arlington, MA) ;
Williams; Anthony; (Melrose, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
38535763 |
Appl. No.: |
11/323261 |
Filed: |
December 30, 2005 |
Current U.S.
Class: |
530/324 |
Current CPC
Class: |
C07K 14/8114 20130101;
C07K 14/8121 20130101 |
Class at
Publication: |
530/324 ;
514/012 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 38/17 20060101 A61K038/17 |
Claims
1. A polypeptide comprising the amino acid sequence: Met His Ser
Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg
Trp Phe Phe Asn Ile Phe Thr Arg Gin Cys Glu Glu Phe Ile Tyr Gly Gly
Cys Glu Gly Asn Gin Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met
Cys Thr Arg Asp (amino acids 3-60 of SEQ ID NO:2), wherein the
polypeptide inhibits kallikrein.
2. The polypeptide of claim 1, wherein the polypeptide comprises
the amino acid sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys
Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile
Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly n Gln
Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp
(SEQ ID NO:2).
3. The polypeptide of claim 1, wherein the polypeptide consists of
the amino acid sequence: Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (amino
acids 3-60 of SEQ ID NO:2).
4. The polypeptide of claim 2, wherein the polypeptide consists of
the amino acid sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys
Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile
Phe Thr Arg Gln Cys Glu Glu Phe Re Tyr Gly Gly Cys Glu Gly Asn Gln
Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp
(SEQ ID NO:2).
5. An isolated polypeptide comprising the amino acid sequence: Met
His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His
Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gin Cys Glu Glu Phe Ile Tyr
Gly Gly Cys Glu Gly Asn Gln Asn Arg: Phe Glu Ser Leu Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp (amino acids 3-60 of SEQ D NO:2),
wherein the polypeptide inhibits kallikrein.
6. The isolated polypeptide of claim 5, wherein the polypeptide
comprises the amino acid sequence: Glu Ala Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe
Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly
Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg
Asp (SEQ ID) NO:2).
7. The isolated polypeptide of claim 5, wherein the polypeptide
consists of the amino acid sequence: Met His Ser Phe Cys Ala Phe
Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn
Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn
Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp
(amino acids 3-60 of SEQ ID NO:2).
8. The isolated polypeptide of claim 6, wherein the polypeptide
consists of the amino acid sequence: Glu Ala Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe
Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu
Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr
Arg Asp (SEQ ID NO:2).
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. application Ser.
No. 10/456,986, filed Jun. 6, 2003, which claims the benefit from
U.S. Provisional Application No. 60/387,239, filed Jun. 7, 2002,
and U.S. Provisional Application No. 60/407,003, filed Aug. 28,
2002.
[0002] The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Proteases are involved in a broad range of biological
pathways. In particular, serine proteases such as kallikrein,
plasmin, elastase, urokinase plasminogen activator, thrombin, human
lipoprotein-associated coagulation inhibitor, and coagulation
factors such as factors VIIa, IXa, Xa, XIa, and XIIa have been
implicated in pathways affecting blood flow, e.g., general and
focal ischemia, tumor invasion, fibrinolysis, perioperative blood
loss, and inflammation. Inhibitors of specific serine proteases,
therefore, have received attention as potential drug targets for
various ischemic maladies.
[0004] One such inhibitor, aprotinin (also called bovine pancreatic
trypsin inhibitor or BPTI), obtained from bovine lung, has been
approved in the United States for prophylactic use in reducing
perioperative blood loss and the need for transfusion in patients
undergoing cardiopulmonary bypass (CPB), e.g., in the course of a
coronary artery bypass grafting procedure. Aprotinin is
commercially available under the trade name TRASYLOL.R.TM. (Bayer
Corporation Pharmaceutical Division, West Haven, Conn.) and was
previously approved for use to treat pancreatitis. The
effectiveness of aprotinin is associated with its relatively
non-specific abilities to inhibit a variety of serine proteases,
including plasma kallikrein and plasmin. These proteases are
important in a number of pathways of the contact activation system
(CAS).
[0005] CAS is initially activated when whole blood contacts the
surface of foreign substrates (e.g., kaolin, glass, dextran
sulfate, or damaged bone surfaces). Kallikrein, a serine protease,
is a plasma enzyme that initiates the CAS cascade leading to
activation of neutrophils, plasmin, coagulation, and various
kinins. Kallikrein is secreted as a zymogen (pre-kallikrein) that
circulates as an inactive molecule until activated by a proteolytic
event early in the contact activation cascade. Clearly, specific
inhibition of kallikrein would be a very attractive approach to
control blood loss associated with CPB and the onset of systemic
inflammatory response (SIR) as would be encountered during, for
example, various invasive surgical procedures.
[0006] Despite being the only licensed compound for preventing
perioperative blood loss in CPB for coronary artery bypass grafting
(CABG) procedures, aprotinin is not as widely used as would be
expected. There are serious concerns regarding the use of this
bovine polypeptide in patients who require CPB, and in particular
the use of this compound in CABG procedures. Aprotinin is not
specific for kallikrein, but interacts with additional enzymes
(e.g., plasmin) in multiple pathways. Thus, the mechanism of action
of aprotinin is largely speculative, and the lack of precise
understanding of what is affected during aprotinin treatment
produces the risk of complications during treatment. One frequently
cited complication is uncontrolled thrombosis, due to aprotinin's
actions upon the fibrinolytic pathway. There is concern not only
over such hyperacute events as major vessel thrombosis in the
perioperative period, but also over graft patency after the CABG
procedure. Furthermore, as a naturally occurring protein obtained
from bovine lung, administration of aprotinin in humans can elicit
severe hypersensitivity or anaphylactic or anaphylactoid reactions
after the first and, more often, after repeat administration to
patients. This is particularly of concern in the large number of
patients who have repeat CABG procedures. In addition, there is an
increasing public concern regarding use of material derived from
bovine sources as a potential vector for the transmission of bovine
spongiform encephalopathy to humans.
[0007] These concerns make clear that a need remains for more
effective and more specific means and methods for preventing or
reducing perioperative blood loss and the onset of SIR in a patient
subjected to surgery resulting in activation of the CAS, such as
CABG procedures in patients of CPB, or hip replacement.
SUMMARY OF THE INVENTION
[0008] This invention is based on the discovery of peptides that
inhibit serine proteases. Serine proteases such as, for example,
kallikrein, are involved in, for example, pathways leading to
excessive perioperative blood loss and the onset of systemic
inflammatory response. Preferred kallikrein peptide inhibitors
include those described in U.S. Pat. Nos. 6,333,402 and 6,057,287
to Markland et al., the contents of which are incorporated herein
by reference in their entirety. The invention is directed in part
to the use of the peptides in therapeutic methods and compositions
suitable for use in eliminating or reducing various ischemias,
including but not limited to perioperative blood loss, and the
onset of systemic inflammatory response. Perioperative blood loss
results from invasive surgical procedures that lead to contact
activation of complement components and the
coagulation/fibrinolysis systems. More specifically, the invention
provides methods of using kallikrein inhibitors to reduce or
prevent perioperative blood loss and a systemic inflammatory
response in patients subjected to invasive surgical procedures,
especially cardiothoracic surgeries.
[0009] In one embodiment, the invention is directed to a method for
preventing or reducing ischemia in a patient comprising
administering to the patient a composition comprising a polypeptide
comprising the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6
Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18
Xaa19 Xaa20Xaa21Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys
Xaa31Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40Xaa41Xaa42 Xaa43
Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50Cys Xaa52 Xaa53 Xaa54 Cys
Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), wherein Xaa1, Xaa2, Xaa3, Xaa4,
Xaa56, Xaa57 or Xaa58 are each individually an amino acid or
absent; Xaa10 is an amino acid selected from the group consisting
of: Asp and Glu; Xaa11 is an amino acid selected from the group
consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 is
an amino acid selected from the group consisting of: Arg, His, Pro,
Asn, Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acid
selected from the group consisting of: Arg, Lys, Ala, Ser, Gly,
Met, Asn and Gln; Xaa16 is an amino acid selected from the group
consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an amino acid
selected from the group consisting of: Ala, Asn, Ser, Ile, Gly,
Val, Gln and Thr; Xaa18 is an amino acid selected from the group
consisting of: His, Leu, Gln and Ala; Xaa19 is an amino acid
selected from the group consisting of: Pro, Gln, Leu, Asn and Ile;
Xaa21 is an amino acid selected from the group consisting of: Trp,
Phe, Tyr, His and Ile; Xaa22 is an amino acid selected from the
group consisting of: Tyr and Phe; Xaa23 is an amino acid selected
from the group consisting of: Tyr and Phe; Xaa31 is an amino acid
selected from the group consisting of: Glu, Asp, Gln, Asn, Ser,
Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected from
the group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser,
Ala, Gly and Val; Xaa34 is an amino acid selected from the group
consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is
an amino acid selected from the group consisting of: Tyr, Trp and
Phe; Xaa39 is an amino acid selected from the group consisting of:
Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid selected from
the group consisting of: Gly and Ala; Xaa43 is an amino acid
selected from the group consisting of: Asn and Gly; Xaa45 is an
amino acid selected from the group consisting of: Phe and Tyr; and
wherein the polypeptide inhibits kallikrein.
[0010] In a particular embodiment, the ischemia is perioperative
blood loss due to a surgical procedure performed on the patient.
The surgical procedure can be a cardiothoracic surgery, such as,
for example, cardiopulmonary bypass or coronary artery bypass
grafting.
[0011] In a particular embodiment, individual amino acid positions
of SEQ ID NO:1 can be one or more of the following: Xaa10 is Asp,
Xaa11 is Asp, Xaa13 is Pro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is
Ala, Xaa18 is His, Xaa19 is Pro, Xaa21 is Trp, Xaa31 is Glu, Xaa32
is Glu, Xaa34 is Ile, Xaa35 is Tyr, Xaa39 is Glu.
[0012] In another embodiment, the invention is directed to a method
for preventing or reducing the onset of systemic inflammatory
response associated with a surgical procedure in a patient
comprising administering to the patient a composition comprising a
polypeptide comprising the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4
Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17
Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28
Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41
Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52
Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), wherein Xaa1,
Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are each individually an
amino acid or absent; Xaa10 is an amino acid selected from the
group consisting of: Asp and Glu; Xaa11 is an amino acid selected
from the group consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and
Thr; Xaa13 is an amino acid selected from the group consisting of:
Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gin; Xaa15 is an
amino acid selected from the group consisting of: Arg, Lys, Ala,
Ser, Gly, Met, Asn and Gin; Xaa16 is an amino acid selected from
the group consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an
amino acid selected from the group consisting of: Ala, Asn, Ser,
Ile, Gly, Val, Gin and Thr; Xaa18 is an amino acid selected from
the group consisting of: His, Leu, Gin and Ala; Xaa19 is an amino
acid selected from the group consisting of: Pro, Gin, Leu, Asn and
lie; Xaa21 is an amino acid selected from the group consisting of:
Trp, Phe, Tyr, His and Ile; Xaa22 is an amino acid selected from
the group consisting of: Tyr and Phe; Xaa23 is an amino acid
selected from the group consisting of: Tyr and Phe; Xaa31 is an
amino acid selected from the group consisting of: Glu, Asp, Gin,
Asn, Ser, Ala, Val, Leu, lie and Thr; Xaa32 is an amino acid
selected from the group consisting of: Glu, Gln, Asp Asn, Pro, Thr,
Leu, Ser, Ala, Gly and Val; Xaa34 is an amino acid selected from
the group consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu;
Xaa35 is an amino acid selected from the group consisting of: Tyr,
Trp and Phe; Xaa39 is an amino acid selected from the group
consisting of: Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid
selected from the group consisting of: Gly and Ala; Xaa43 is an
amino acid selected from the group consisting of: Asn and Gly;
Xaa45 is an amino acid selected from the group consisting of: Phe
and Tyr; and wherein the polypeptide inhibits kallikrein. In a
particular embodiment, the surgical procedure can be a
cardiothoracic surgery, such as, for example, cardiopulmonary
bypass or coronary artery bypass grafting. In a particular
embodiment, individual amino acid positions of SEQ ID NO:1 can be
one or more of the following: Xaa10 is Asp, Xaa11 is Asp, Xaa13 is
Pro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His, Xaa19
is Pro, Xaa21 is Trp, Xaa31 is Glu, Xaa32 is Glu, Xaa34 is Ile,
Xaa35 is Tyr, Xaa39 is Glu.
[0013] In yet another embodiment, the invention is directed to a
method for preventing or reducing the onset of systemic
inflammatory response associated with a surgical procedure in a
patient comprising administering to the patient a composition
comprising a polypeptide consisting of the amino acid sequence: Glu
Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala
Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu
Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2), wherein the
polypeptide inhibits kallikrein. In one embodiment, the surgical
procedure is a cardiothoracic surgery, such as, for example,
cardiopulmonary bypass or coronary artery bypass grafting.
[0014] In another embodiment, the invention is directed to a method
for preventing or reducing ischemia in a patient comprising
administering to the patient a composition comprising a polypeptide
consisting of the amino acid sequence: Glu Ala Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe
Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu
Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr
Arg Asp (SEQ ID NO:2), wherein the polypeptide inhibits kallikrein.
In a particular embodiment, the ischemia can be perioperative blood
loss due to a surgical procedure performed on the patient. In one
embodiment, the surgical procedure is a cardiothoracic surgery,
such as, for example, cardiopulmonary bypass or coronary artery
bypass grafting.
[0015] In yet another embodiment, the invention is directed to a
method for preventing or reducing the onset of systemic
inflammatory response associated with a surgical procedure in a
patient comprising administering to the patient a composition
comprising a polypeptide consisting of the amino acid sequence: Met
His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His
Pro Arg Trp Phe Phe Asn lie Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr
Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys
Lys Met Cys Thr Arg Asp (amino acids 3-60 of SEQ ID NO:2), wherein
the polypeptide inhibits kallikrein. In one embodiment, the
surgical procedure is a cardiothoracic surgery, such as, for
example, cardiopulmonary bypass or coronary artery bypass
grafting.
[0016] In another embodiment, the invention is directed to a method
for preventing or reducing ischemia in a patient comprising
administering to the patient a composition comprising a polypeptide
consisting of the amino acid sequence: Met His Ser Phe Cys Ala Phe
Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn
Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn
Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp
(amino acids 3-60 of SEQ ID NO:2), wherein the polypeptide inhibits
kallikrein. In a particular embodiment, the ischemia can be
perioperative blood loss due to a surgical procedure performed on
the patient. In one embodiment, the surgical procedure is a
cardiothoracic surgery, such as, for example, cardiopulmonary
bypass or coronary artery bypass grafting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a simplified diagram of major multiple pathways
and related events involved in the contact activation system and
systemic inflammatory response (SIR) that can arise in a patient
subjected to soft and bone tissue trauma such as that associated
with a coronary artery bypass grafting (CABG) procedure, especially
when the CABG procedure involves extra-corporeal blood circulation,
such as cardiopulmonary bypass (Bypass Apparatus). Arrows indicate
activation from one component or event to another component or
event in the cascade. Arrows in both directions indicate activating
effects of components or events in both directions. Broken arrows
indicate likely participation of one component or event in the
activation of another component or event. Abbreviations are as
follows: "tPA"=tissue plasminogen activator; "C5a"=a protein
component of the complement system; "fXIIa"=activator protein of
prekallikrein to form active kallikrein; "Extrinsic"=extrinsic
coagulation system; "Intrinsic"=intrinsic coagulation system.
[0018] FIG. 2 shows a portion of a DNA and corresponding deduced
amino acid for a KI polypeptide of the invention in plasmid
pPIC-K503. The inserted DNA encodes the mat.alpha. prepro signal
peptide of Saccharomyces cerevisiae (underlined) fused in frame to
the amino terminus of the PEP-1 KI polypeptide having the amino
acid sequence enclosed by the boxed area. The amino acid sequence
of the PEP-1 KI polypeptide shown in the boxed region is SEQ ID
NO:2, and the corresponding nucleotide coding sequence of the KI
polypeptide is SEQ ID NO:3. The dashed arrows indicate the location
and direction of two PCR primer sequences in AOX regions that were
used to produce sequencing templates. DNA sequence for the entire
nucleotide sequence of the figure comprises the structural coding
sequence for the fusion protein and is designated SEQ ID NO:27. The
entire amino acid sequence is SEQ ID NO:28. The double underlined
portion of the sequence indicates a diagnostic probe sequence.
BstBI and EcoRI indicate locations of their respective palindromic,
hexameric, restriction endonuclease sites in the sequence.
Asterisks denote translational stop codons.
[0019] FIGS. 3A and 3B show an alignment of amino acid sequences of
the preferred embodiments of the invention, the native LACI
sequence from which these variants were derived (SEQ ID NO:32), and
other known Kunitz domains (SEQ ID NOS:29-31 and 33-53). Cysteine
residues are highlighted.
DETAILED DESCRIPTION OF THE INVENTION
[0020] A description of preferred embodiments of the invention
follows.
[0021] The invention is based on the discovery of a group of
kallikrein inhibitor (KI) polypeptides that inhibit plasma
kallikrein with a specificity that permits their use in improved
methods of preventing or reducing ischemia such as, for example,
perioperative blood loss and/or a systemic inflammatory response
(SIR) induced by kallikrein, especially, for example, in patients
undergoing surgical procedures and particularly surgical procedures
involving cardiothoracic surgery, e.g., cardiopulmonary bypass
(CPB), such as a coronary artery bypass graft (CABG) procedures.
K's can be used specifically for, e.g., pediatric cardiac surgery,
lung transplantation, total hip replacement and orthotopic liver
transplantation, and to reduce or prevent perioperative stroke
during CABG, extracorporeal membrane oxygenation (ECMO) and
cerebrovascular accidents (CVA) during these procedures.
[0022] Cardiothoracic surgery is surgery of the chest area, most
commonly the heart and lungs. Typical diseases treated by
cardiothoracic surgery include coronary artery disease, tumors and
cancers of the lung, esophagus and chest wall, heart vessel and
valve abnormalities, and birth defects involving the chest or
heart. Where cardiothoracic surgery is utilized for treatment, the
risk of blood loss (e.g., surgery-induced ischemia) and the onset
of a systemic inflammatory response (SIR) is incurred.
Surgery-induced SIR can result in severe organ dysfunction
(systemic inflammatory response syndrome; SIRS).
Polypeptides Useful in the Invention
[0023] KI polypeptides useful in the invention comprise Kunitz
domain polypeptides. In one embodiment these Kunitz domains are
variant forms of the looped structure comprising Kunitz domain 1 of
human lipoprotein-associated coagulation inhibitor (LACI) protein.
LACI contains three internal, well-defined, peptide loop structures
that are paradigm Kunitz domains (Girard, T. et al., 1989. Nature,
338:518-520). The three Kunitz domains of LACI confer the ability
to bind and inhibit kallikrein, although not with exceptional
affinity. Variants of Kunitz domain 1 of LACI described herein have
been screened, isolated and bind kallikrein with enhanced affinity
and specificity (see, for example, U.S. Pat. Nos. 5,795,865 and
6,057,287, incorporated herein by reference). An example of a
preferred polypeptide useful in the invention has the amino acid
sequence defined by amino acids 3-60 of SEQ ID NO:2.
[0024] Every polypeptide useful in the invention binds kallikrein,
and preferred polypeptides are also kallikrein inhibitors (KI) as
determined using kallikrein binding and inhibition assays known in
the art. The enhanced affinity and specificity for kallikrein of
the variant Kunitz domain polypeptides described herein provides
the basis for their use in cardiothoracic surgery, e.g., CPB and
especially CABG surgical procedures, to prevent or reduce
perioperative blood loss and/or the onset of SIR in patients
undergoing such procedures. The KI polypeptides used in the
invention have or comprise the amino acid sequence of a variant
Kunitz domain polypeptide originally isolated by screening phage
display libraries for the ability to bind kallikrein.
[0025] KI polypeptides useful in the methods and compositions of
the invention comprise a Kunitz domain polypeptide comprising the
amino acid sequence: TABLE-US-00001 Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6
Xaa7 (SEQ ID NO:1) Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16
Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27
Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40
Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys
Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58
[0026] "Xaa" refers to a position in a peptide chain that can be
any of a number of different amino acids. For example, for the KI
peptides described herein, Xaa10 can be Asp or Glu; Xaa11 can be
Asp, Gly, Ser, Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg,
His, Asn, Ser, Thr, Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys,
Ala, Ser, Gly, Met, Asn or Gln; Xaa16 can be Ala, Gly, Ser, Asp or
Asn; Xaa17 can be Ala, Asn, Ser, Ile, Gly, Val, Gln or Thr; Xaa18
can be His, Leu, Gln or Ala; Xaa19 can be Pro, Gln, Leu, Asn or
Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile; Xaa31 can be Glu, Asp,
Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32 can be Glu, Gln,
Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 can be Ile,
Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp or Phe;
Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,
Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,
Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and
Xaa54 can be any amino acid. Additionally, each of the first four
and at last three amino acids of SEQ ID NO:1 can optionally be
present or absent and can be any amino acid, if present.
[0027] Peptides defined according to SEQ ID NO:1 form a set of
polypeptides that bind to kallikrein. For example, in a preferred
embodiment of the invention, a KI polypeptide useful in the methods
and compositions of the invention has the following variable
positions: Xaa11 can be Asp, Gly, Ser or Val; Xaa13 can be Pro,
Arg, His or Asn; Xaa15 can be Arg or Lys; Xaa16 can be Ala or Gly;
Xaa17 can be Ala, Asn, Ser or Ile; Xaa18 can be His, Leu or Gln;
Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trp or Phe; Xaa31 is
Glu; Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr or Ser; Xaa35
is Tyr; and Xaa39 can be Glu, Gly or Ala.
[0028] A more specific embodiment of the claimed invention is
defined by the following amino acids at variable positions: Xaa10
is Asp; Xaa11 is Asp; Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16
can be Ala or Gly; Xaa17 is Ala; Xaa18 is His; Xaa19 is Pro; Xaa21
is Trp; Xaa31 is Glu; Xaa32 is Glu; Xaa34 can be Ile or Ser; Xaa35
is Tyr; and Xaa39 is Gly.
[0029] Also encompassed within the scope of the invention are
peptides that comprise portions of the polypeptides described
herein. For example, polypeptides could comprise binding domains
for specific kallikrein epitopes. Such fragments of the
polypeptides described herein would also be encompassed.
[0030] KI polypeptides useful in the methods and compositions
described herein comprise a Kunitz domain. A subset of the
sequences encompassed by SEQ ID NO:1 are described by the following
(where not indicated, "Xaa" refers to the same set of amino acids
that are allowed for SEQ ID NO:1): TABLE-US-00002 (SEQ ID NO:54)
Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11 Gly Xaa13 Cys Xaa15
Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn Ile Phe Thr Arg Gln
Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Gly Asn Gln Asn
Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp. (amino
acids 3-60 of SEQ ID NO:2) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:4) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys
Ala Asn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:5) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Thr Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO:6) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Gln Phe Thr Tyr Gly Gly Cys Ala Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:7) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys
Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:8) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO:9) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly His Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:10) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys
Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:11) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Gly Ala His Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO:12) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:13) Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg
Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:14) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala Gln Pro Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO:15) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly Ser Cys Arg Ala Ala His Leu Arg Trp Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:16) Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Ser Cys Arg
Ala Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:17) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO:18) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Asp Gly His Cys Arg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:19) Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg
Gly Asn Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:20) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg Gly Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO:21) Met His Ser Phe Cys Ala Phe Lys Ala Asp
Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe Asn Ile Phe Thr
Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg
Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID
NO:22) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Arg
Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp.
[0031] FIGS. 3A and 3B provides an amino acid sequence alignment of
these sequences, the native LACI sequence from which these variants
were derived (SEQ ID NO:32), and other known Kunitz domains (SEQ ID
NOS: 29-31 and 33-53).
[0032] The KI polypeptides useful in the methods and compositions
described herein can be made synthetically using any standard
polypeptide synthesis protocol and equipment. For example, the
stepwise synthesis of a KI polypeptide described herein can be
carried out by the removal of an amino (N) terminal-protecting
group from an initial (i.e., carboxy-terminal) amino acid, and
coupling thereto of the carboxyl end of the next amino acid in the
sequence of the polypeptide. This amino acid is also suitably
protected. The carboxyl group of the incoming amino acid can be
activated to react with the N-terminus of the bound amino acid by
formation into a reactive group such as formation into a
carbodiimide, a symmetric acid anhydride, or an "active ester"
group such as hydroxybenzotriazole or pentafluorophenyl esters.
Preferred solid-phase peptide synthesis methods include the BOC
method, which utilizes tert-butyloxycarbonyl as the .alpha.-amino
protecting group, and the FMOC method, which utilizes
9-fluorenylmethloxycarbonyl to protect the .alpha.-amino of the
amino acid residues. Both methods are well known to those of skill
in the art (Stewart, J. and Young, J., Solid-Phase Peptide
Synthesis (W. H. Freeman Co., San Francisco 1989); Merrifield, J.,
1963. Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky,
A., The Practice of Peptide Synthesis (Springer-Verlag, New York
1984), the entire teachings of these references is incorporated
herein by reference). If desired, additional amino- and/or
carboxy-terminal amino acids can be designed into the amino acid
sequence and added during polypeptide synthesis.
[0033] Alternatively, Kunitz domain polypeptides and KI
polypeptides useful in the compositions and methods of the
invention can be produced by recombinant methods using any of a
number of cells and corresponding expression vectors, including but
not limited to bacterial expression vectors, yeast expression
vectors, baculovirus expression vectors, mammalian viral expression
vectors, and the like. Kunitz domain polypeptides and KI
polypeptides useful in the compositions and methods of the
invention can also be produced transgenically using nucleic acid
molecules comprising a coding sequence for a Kunitz domain or KI
polypeptide described herein, wherein the nucleic acid molecule can
be integrated into and expressed from the genome of a host animal
using transgenic methods available in the art. In some cases, it
could be necessary or advantageous to fuse the coding sequence for
a Kunitz domain polypeptide or a KI polypeptide comprising the
Kunitz domain to another coding sequence in an expression vector to
form a fusion polypeptide that is readily expressed in a host cell.
Preferably, the host cell that expresses such a fusion polypeptide
also processes the fusion polypeptide to yield a Kunitz domain or
KI polypeptide useful in the invention that contains only the
desired amino acid sequence. Obviously, if any other amino acid(s)
remain attached to the expressed Kunitz domain or KI polypeptide,
such additional amino acid(s) should not diminish the kallikrein
binding and/or kallikrein inhibitory activity of the Kunitz domain
or KI polypeptide so as to preclude use of the polypeptide in the
methods or compositions of the invention.
[0034] A preferred recombinant expression system for producing KI
polypeptides useful in the methods and compositions described
herein is a yeast expression vector, which permits a nucleic acid
sequence encoding the amino acid sequence for a KI polypeptide or
Kunitz domain polypeptide to be linked in the same reading frame
with a nucleotide sequence encoding the mat.alpha. prepro leader
peptide sequence of Saccharomyces cerevisiae, which in turn is
under the control of an operable yeast promoter. The resulting
recombinant yeast expression plasmid can then be transformed by
standard methods into the cells of an appropriate, compatible yeast
host, which cells are able to express the recombinant protein from
the recombinant yeast expression vector. Preferably, a host yeast
cell transformed with such a recombinant expression vector is also
able to process the fusion protein to provide an active KI
polypeptide useful in the methods and compositions of the
invention. A preferred yeast host for producing recombinant Kunitz
domain polypeptides and KI polypeptides comprising such Kunitz
domains is Pichia pastoris.
[0035] As noted above, KI polypeptides that are useful in the
methods and compositions described herein can comprise a Kunitz
domain polypeptide described herein. Some KI polypeptides can
comprise an additional flanking sequence, preferably of one to six
amino acids in length, at the amino and/or carboxy-terminal end,
provided such additional amino acids do not significantly diminish
kallikrein binding affinity or kallikrein inhibition activity so as
to preclude use in the methods and compositions described herein.
Such additional amino acids can be deliberately added to express a
KI polypeptide in a particular recombinant host cell or can be
added to provide an additional function, e.g., to provide a peptide
to link the KI polypeptide to another molecule or to provide an
affinity moiety that facilitates purification of the polypeptide.
Preferably, the additional amino acid(s) do not include cysteine,
which could interfere with the disulfide bonds of the Kunitz
domain.
[0036] An example of a preferred Kunitz domain polypeptide useful
in the methods and compositions of the invention has the amino acid
sequence of residues 3-60 of SEQ ID NO:2. When expressed and
processed in a yeast fusion protein expression system (e.g., based
on the integrating expression plasmid pHIL-D2), such a Kunitz
domain polypeptide retains an additional amino terminal Glu-Ala
dipeptide from the fusion with the mat.alpha. prepro leader peptide
sequence of S. cerevisiae. When secreted from the yeast host cell,
most of the leader peptide is processed from the fusion protein to
yield a functional KI polypeptide (referred to herein as "PEP-1")
having the amino acid sequence of SEQ ID NO:2 (see boxed region in
FIG. 2).
[0037] Particularly preferred KI polypeptides useful in the methods
and compositions described herein have a binding affinity for
kallikrein that is on the order of 1000 times higher than that of
aprotinin, which is currently approved for use in CABG procedures
to reduce blood loss. The surprisingly high binding affinities of
such KI polypeptides described herein indicate that such KI
polypeptides exhibit a high degree of specificity for kallikrein to
the exclusion of other molecular targets (see Table 1, below).
Thus, use of such polypeptides according to the invention reduces
much of the speculation as to the possible therapeutic targets in a
patient. The lower degree of specificity exhibited by, for example,
aprotinin, leads to possible pleiotropic side effects and ambiguity
as to its therapeutic mechanism.
[0038] The polypeptides defined by, for example, SEQ ID NO:1
contain invariant positions, e.g., positions 5, 14, 30, 51 and 55
can be Cys only. Other positions such as, for example, positions 6,
7, 8, 9, 20, 24, 25, 26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49,
50, 52, 53 and 54 can be any amino acid (including non-naturally
occurring amino acids). In a particularly preferred embodiment, one
or more amino acids correspond to that of a native sequence (e.g.,
SEQ ID NO:32, see FIG. 3). In a preferred embodiment, at least one
variable position is different from that of the native sequence. In
yet another preferred embodiment, the amino acids can each be
individually or collectively substituted by a conservative or
non-conservative amino acid substitution. Conservative amino acid
substitutions replace an amino acid with another amino acid of
similar chemical structure and may have no affect on protein
function. Non-conservative amino acid substitutions replace an
amino acid with another amino acid of dissimilar chemical
structure. Examples of conserved amino acid substitutions include,
for example, Asn->Asp, Arg->Lys and Ser->Thr. In a
preferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 and/or 21 of these amino acids can be
independently or collectively, in any combination, selected to
correspond to the corresponding position of SEQ ID NO:2.
[0039] Other positions, for example, positions 10, 11, 13, 15, 16,
17, 18, 19, 21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, can be
any of a selected set of amino acids. Thus SEQ ID NO:1 defines a
set of possible sequences. Each member of this set contains, for
example, a cysteine at positions 5, 14, 30, 51 and 55, and any one
of a specific set of amino acids at positions 10, 11, 13, 15, 16,
17, 18, 19, 221, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45. In a
preferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18 and/or 19 of these amino acids can be
independently or collectively, in any combination, selected to
correspond to the corresponding position of SEQ ID NO:2. The
peptide preferably has at least 80%, at least 85%, at least 90% or
at least 95% identity to SEQ ID NO:2.
Methods and Compositions
[0040] The present invention is also directed to methods for
preventing or reducing ischemia. Preferred in the invention are
methods for preventing or reducing perioperative blood loss and/or
a systemic inflammatory response (SIR) in a patient, especially
associated with cardiothoracic surgery. A method for treatment
involves the administration of a KI polypeptide comprising a Kunitz
domain. One embodiment of the method involves using a peptide
containing an amino acid sequence of SEQ ID NO:1 that has an
affinity for kallikrein that is approximately 1000-fold or more
higher than that of a broad range serine protease, e.g., aprotinin,
which is isolated from bovine lung and currently approved for use
in CABG procedures (TRASYLOL.R.TM., Bayer Corporation
Pharmaceutical Division, West Haven, Conn.).
[0041] Patients subjected to any of a number of surgical
procedures, especially those involving extra-corporeal circulation,
e.g., cardiothoracic surgery, such as, for example, CPB, and/or
bone trauma, such as sternal split or hip replacement, are at risk
for perioperative blood loss and inflammation. Contact of a
patient's blood with the cut surfaces of bone or of CPB equipment
is sufficient to activate one or several undesirable cascade
responses, including a contact activation system (CAS), which can
lead to extensive perioperative blood loss requiring immediate
blood transfusion, as well as a systemic inflammatory response
(SIR), which, in turn, can result in permanent damage to tissues
and organs. While not desiring to be limited to any particular
mechanism or theory, it appears that the blood loss that occurs
associated with cardiothoracic surgery, e.g., CPB, as in a CABG
procedure, probably results from extensive capillary leakage, which
can result in significant loss of blood that must be replaced by
immediate blood transfusion.
[0042] The methods described herein are useful for preventing or
reducing various ischemias including, for example, perioperative
blood loss and SIR in a patient subjected to a surgical procedure,
and especially wherein the surgical procedure requires
extra-corporeal circulation,, e.g., cardiothoracic surgery, such
as, for example, CPB. The methods of the invention are particularly
useful for preventing or reducing perioperative blood loss and/or
SIR in a patient subjected to a CABG procedure requiring CPB or
other cardiac surgery.
[0043] Preferred compositions for medical use comprise a KI
polypeptide described herein. Such compositions useful can further
comprise one or more pharmaceutically acceptable buffers, carriers,
and excipients, which can provide a desirable feature to the
composition including, but not limited to, enhanced administration
of the composition to a patient, enhanced circulating half-life of
the KI polypeptide of the composition, enhanced compatibility of
the composition with patient blood chemistry, enhanced storage of
the composition, and/or enhanced efficacy of the composition upon
administration to a patient. In addition to a KI polypeptide
described herein, compositions can further comprise one or more
other pharmaceutically active compounds that provide an additional
prophylactic or therapeutic benefit to a patient of an invasive
surgical procedure.
[0044] Compositions useful in the methods of the invention comprise
any of the Kunitz domain polypeptides or KI polypeptides comprising
such Kunitz domain polypeptides described herein. Particularly
preferred are KI polypeptides comprising a Kunitz domain
polypeptide having a 58-amino acid sequence of amino acids 3-60 of
SEQ ID NO:2. An example of such a particularly preferred KI
polypeptide useful in the methods and compositions of the invention
is the PEP-1 KI polypeptide having the 60-amino acid sequence of
SEQ ID NO:2. A nucleotide sequence encoding the amino acid sequence
of SEQ ID NO:2 is provided in SEQ ID NO:3 (see, e.g., nucleotides
309-488 in FIG. 2). It is understood that based on the known
genetic code, the invention also provides degenerate forms of the
nucleotide sequence of SEQ ID NO:3 by simply substituting one or
more of the known degenerate codons for each amino acid encoded by
the nucleotide sequence. Nucleotides 7-180 of SEQ ID NO:3, and
degenerate forms thereof, encode the non-naturally occurring Kunitz
domain polypeptide having the 58-amino acid sequence of amino acids
3-60 of SEQ ID NO:2.
[0045] Any of a variety of nucleic acid molecules can comprise the
nucleotide sequence of nucleotides 7-180 of SEQ ID NO:3, degenerate
forms, and portions thereof, including but not limited to,
recombinant phage genomes, recombinant mammalian viral vectors,
recombinant insect viral vectors, yeast mini chromosomes, and
various plasmids. Such plasmids include those used to clone and/or
express such nucleotide coding sequences. Expression vectors
provide a promoter, which can be operably linked to a particular
nucleotide sequence and an appropriate host cell, which is able to
transcribe the particular nucleotide coding sequence into a
functional messenger RNA (mRNA) and also translate the mRNA into
the corresponding polypeptide. A polypeptide so produced can then
be isolated from the host cell. Nucleic acid molecules comprising a
nucleic acid sequence encoding a Kunitz domain or KI polypeptide
described herein can be made by standard nucleic acid synthesis
methods, recombinant DNA methodologies, polymerase chain reaction
(PCR) methods, and any combination thereof.
Perioperative Blood Loss and Reduced Heart Bloodflow
[0046] Due to the many advances in medicine, a number of highly
invasive surgical procedures are carried out each day that result
in blood loss, or place patients at a high risk for blood loss.
Such patients must be carefully monitored to restore and maintain
normal blood supply and hemostasis, and they may need blood
transfusions. Surgical procedures that involve blood loss include
those involving extra-corporeal circulation methods such as
cardiothoracic surgery, e.g., CPB. In such methods, a patient's
heart is stopped and the circulation, oxygenation, and maintenance
of blood volume are carried out artificially using an
extra-corporeal circuit and a synthetic membrane oxygenator. These
techniques are commonly used during cardiac surgery. Additionally,
it is apparent that surgery involving extensive trauma to bone,
such as the sternal split necessary in CABG or hip replacement
procedures, is also associated with activation of the CAS, which
can result in a variety of disruptions in the blood and
vasculature.
[0047] Atherosclerotic coronary artery disease (CAD) causes a
narrowing of the lumen of one or several of the coronary arteries;
this limits the flow of blood to the myocardium (i.e., the heart
muscle) and can cause angina, heart failure, and myocardial
infarcts. In the end stage of coronary artery atherosclerosis, the
coronary circulation can be almost completely occluded, causing
life threatening angina or heart failure, with a very high
mortality. CABG procedures may be required to bridge the occluded
blood vessel and restore blood to the heart; these are potentially
life saving. CABG procedures are among the most invasive of
surgeries in which one or more healthy veins or arteries are
implanted to provide a "bypass" around the occluded area of the
diseased vessel. CABG procedures carry with them a small but
important perioperative risk, but they are very successful in
providing patients with immediate relief from the mortality and
morbidity of atherosclerotic cardiovascular disease. Despite these
very encouraging results, repeat CABG procedures are frequently
necessary, as indicated by a clear increase in the number of
patients who eventually undergo second and even third procedures;
the perioperative mortality and morbidity seen in primary CABG
procedures is increased in these re-do procedures.
[0048] There have been improvements in minimally invasive surgical
techniques for uncomplicated CAD. However, nearly all CABG
procedures performed for valvular and/or congenital heart disease,
heart transplantation, and major aortic procedures, are still
carried out on patients supported by CPB. In CPB, large cannulae
are inserted into the great vessels of a patient to permit
mechanical pumping and oxygenation of the blood using a membrane
oxygenator. The blood is returned to the patient without flowing
through the lungs, which are hypoperfused during this procedure.
The heart is stopped using a cardioplegic solution, the patient
cooled to help prevent brain damage, and the peripheral circulating
volume increased by an extracorporeal circuit, i.e., the CPB
circuit, which requires "priming" with donor blood and saline
mixtures are used to fill the extracorporeal circuit. CPB has been
extensively used in a variety of procedures performed for nearly
half a century with successful outcomes. The interaction between
artificial surfaces, blood cells, blood proteins, damaged vascular
endothelium, and extravascular tissues, such as bone, disturbs
hemostasis and frequently activates the CAS, which, as noted above,
can result in a variety of disruptions in the blood and
vasculature. Such disruption leads to excess perioperative
bleeding, which then requires immediate blood transfusion. A
consequence of circulating whole blood through an extracorporeal
circuit in CPB can also include the systemic inflammatory response
(SIR), which is initiated by contact activation of the coagulation
and complement systems. Indeed, much of the morbidity and mortality
associated with seemingly mechanically successful CPB surgical
procedures is the result of the effects of activating coagulation,
fibrinolysis, or complement systems. Such activation can damage the
pulmonary system, leading to adult respiratory distress syndrome
(ARDS), impairment of kidney and splanchnic circulation, and
induction of a general coagulopathy leading to blood loss and the
need for transfusions. In addition to the dangers of perioperative
blood loss, additional pathologies associated with SIR include
neurocognitive deficits, stroke, renal failure, acute myocardial
infarct, and cardiac tissue damage.
[0049] Blood transfusions also present a significant risk of
infection and elevate the cost of CABG or other similar procedures
that require CPB. In the absence of any pharmacological
intervention, three to seven units of blood must typically be
expended on a patient, even with excellent surgical techniques.
Accordingly, there is considerable incentive for the development of
new and improved pharmacologically effective compounds to reduce or
prevent perioperative bleeding and SIR in patients subjected to CPB
and CABG procedures.
Administration and Dosing Considerations for KI Polypeptides
[0050] KI polypeptides described herein can be administered to a
patient before, during, and/or after a surgical procedure in a
pharmaceutically acceptable composition. The term "pharmaceutically
acceptable" composition refers to a non-toxic carrier or excipient
that may be administered to a patient, together with a compound of
this invention, and wherein the carrier or excipient not destroy
the biological or pharmacological activity of the composition. KI
polypeptides described herein can be administered locally or
systemically by any suitable means for delivery of a kallikrein
inhibitory amount of the KI polypeptides to a patient including but
not limited to systemic administrations such as, for example,
intravenous and inhalation. Parenteral administration is
particularly preferred.
[0051] For parenteral administration, the polypeptides can be
injected intravenously, intramuscularly, intraperitoneally, or
subcutaneously. Intravenous adminsistration is preferred.
Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. Other
pharmaceutically acceptable carriers include, but are not limited
to, sterile water, saline solution, and buffered saline (including
buffers like phosphate or acetate), alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, paraffin, etc. Where necessary, the
composition can also include a solubilizing agent and a local
anaesthetic such as lidocaine to ease pain at the site of the
injection, preservatives, stabilizers, wetting agents, emulsifiers,
salts, lubricants, etc. as long as they do not react deleteriously
with the active compounds. Similarly, the composition can comprise
conventional excipients, e.g., pharmaceutically acceptable organic
or inorganic carrier substances suitable for parenteral, enteral or
intranasal application which do not deleteriously react with the
active compounds. Generally, the ingredients will be supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent in activity units. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade "water for injection" or saline. Where the composition is to
be administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients can be
mixed prior to administration.
[0052] Preferably, the methods of the invention comprise
administering a KI polypeptide to a patient as an intravenous
infusion according to any approved procedure. Thus, a KI
polypeptide described herein can be administered to a patient
subjected to a CABG procedure at the times similar to those
currently used in approved protocols for administering aprotinin
and in an amount necessary to provide a patient with a required
number or concentration of kallikrein inhibitory units (KIU).
According to the invention, a KI polypeptide described herein can
also be administered to a patient in the immediate postoperative
period, when bleeding abnormalities can occur as a consequence of
downstream effects of SIR. For example, in a procedure involving
CPB, a KI polypeptide described herein can be administered to a
patient as an initial loading dose, e.g., an effective amount over
the course of a convenient time, such as 10 minutes, prior to
induction of anesthesia. Then, at induction of anesthesia, a second
dose of KI polypeptide can be injected into the CPB priming fluid
("pump prime volume"). The patient can then be placed on a
continuous and controlled intravenous infusion dose for the
duration of the surgical procedure, and after the procedure if
indicated.
[0053] Currently there are two regimens approved in the United
States for administering aprotinin to a patient undergoing a CABG
procedure (see, product label and insert for TRASYLOL.R.TM., Bayer
Corporation Pharmaceutical Division, West Haven, Conn.). One such
approved regimen uses a 2 million KIU intravenous loading dose, 2
million KIU into the pump prime volume, and 500,000 KIU per hour of
surgery. Another approved regimen uses 1 million KIU intravenous
loading dose, 1 million KIU into the pump prime volume, and 250,000
KIU per hour of surgery. As these regimens are based on KIU, the
regimens are readily adapted to any KI polypeptide described herein
once the specific activity and KIU of a particular KI polypeptide
has been determined by standard assays. Owing to the enhanced
binding affinity and inhibitory activity in representative KI
polypeptides described herein relative to aprotinin, it is expected
that such compositions and methods of the invention are likely to
require fewer milligrams (mg) per patient to provide a patient with
the required number or concentration of KIU.
[0054] Several considerations regarding dosing with a KI
polypeptide in methods of the invention can be illustrated by way
of example with the representative PEP-1 KI polypeptide of the
invention having the amino sequence of SEQ ID NO:2 (molecular
weight of 7,054 Daltons).
[0055] Table 1, below, provides a comparison of the affinity
(K.sub.i,app) of the PEP-1 KI polypeptide for kallikrein and eleven
other known plasma proteases.
[0056] 1TABLE 1 Aprotinin Protease Substrate PEP-1 K.sub.i,app (pM)
K.sub.i,app (pM) human plasma kallikrein 44 3.0.times. 10.sup.4
human urine kallikrein>1.times. 10.sup.8 4.0.times. 10.sup.3
porcine pancreatic kallikrein 2.7.times. 10.sup.7 550 human C1r,
activated>2.0.times. 10.sup.8>1.0.times. 10.sup.7 human C1s,
activated>2.0.times. 10.sup.7>1.0.times. 10.sup.8 human
plasma factor XIa 1.0.times. 10.sup.4 ND human plasma factor
XIIa>2.0.times. 10.sup.7>1.0.times. 10.sup.8 human plasmin
1.4.times. 10.sup.5 894 human pancreatic trypsin>2.times.
10.sup.7 ND human pancreatic chymotrypsin>2.0.times. 10.sup.7
7.3.times. 10.sup.5 human neutrophil elastase>2.0.times.
10.sup.7 1.7.times. 10.sup.6 human plasma thrombin>2.0.times.
10.sup.7>1.0.times. 10.sup.8 ND=not determined
[0057] Clearly, the PEP-1 KI polypeptide is highly specific for
human plasma kallikrein. Furthermore, the affinity (K.sub.i,app) of
PEP-1 for kallikrein is 1000 times higher than the affinity of
aprotinin for kallikrein: the K.sub.i,app of PEP-1 for kallikrein
is about 44 pM (Table 1), whereas the K.sub.i,app of aprotinin for
kallikrein is 30,000 pM. Thus, a dose of PEP-1 could be
approximately 1000 times lower than that used for aprotinin on a
per mole basis. However, consideration of several other factors may
provide a more accurate estimation of the dose of PEP-1 required in
practice. Such factors include the amount of kallikrein activated
during CPB in a particular patient, the concentration of kallikrein
required to elicit an SIR, and the bioavailability and
pharmacological distribution of PEP-1 in a patient. Nevertheless,
use of a KI polypeptide in methods according to the invention and
provided in doses currently approved for the use of aprotinin is
still expected to provide significant improvements over the current
use of the less specific, lower affinity, bovine aprotinin.
[0058] For example, the total amount of circulating prekallikrein
in plasma is estimated at approximately 500 nM (Silverberg, M. et
al., "The Contact System and Its Disorders," in Blood: Principles
and Practice of Hematology, Handin, R. et al., eds., J B Lippincott
Co., Philadelphia, 1995). If all of the prekallikrein were
activated, then at least 500 nM of PEP-1 would be required for a
stoichiometric inhibition of kallikrein. An individual having 5
liters of plasma would therefore require about 18 mg of PEP-1 to
achieve a plasma concentration of 500 nM.
[0059] Another factor to consider is the threshold concentration of
kallikrein required to induce a SIR in a patient. If the
concentration of active kallikrein must be maintained below, e.g.,
1 nM, then owing to its high affinity for kallikrein, PEP-1 offers
a significant advantage over aprotinin in the amount of protein
that would be required to inhibit SIR. In particular, a
concentration of PEP-1 of 1 nM would inhibit 99.6% of kallikrein
present at 1 nM (i.e., only 0.4 pM free kallikrein remaining in the
blood), whereas, an aprotinin concentration of 1 nM would only
inhibit 24.5% of the kallikrein present at 1 nM. For aprotinin to
inhibit 99% of the kallikrein at 1 nM, an aprotinin concentration
in the plasma of at least 3.mu.M is required (i.e., 3000 times
higher concentration than for PEP-1).
[0060] For a patient undergoing CPB, an initial clinical dose of
PEP-1 can be estimated from a recommended dose regimen of aprotinin
(.times. 10.sup.6 KIU) mentioned above. Aprotinin is reported in a
package insert to have as specific inhibitory activity of 7143
KIU/mg determined using a dog blood pressure assay. Therefore,
1.times. 10.sup.6 KIU of aprotinin is equivalent to 140 mg of
aprotinin (i.e., 1.times. 10.sup.6 KIU/7143 KIU/mg=140 mg of
aprotinin). In a patient having a blood plasma volume of 5 liters,
140 mg corresponds to approximately 4.3.mu.M aprotinin (molecular
weight of aprotinin is 6512 Daltons). The specific activity of
aprotinin in the standard inhibitory assay used for PEP-1 is 0.4
KIU/mg of polypeptide. A dose of 140 mg would correspond to a
loading dose for aprotinin of 56 KIU (140 mg.times.0.4 KIU/mg=56
KIU). In contrast, since the specific activity of the PEP-1 KI
polypeptide is 10 KIU/mg in the standard inhibition assay, a dose
of only 5.6 mg of PEP-1 would be required to provide the number of
KIUs equivalent to 140 mg of aprotinin. In a patient with a plasma
volume of 5 liters, this corresponds to about 160 nM PEP-1
(molecular weight of PEP-1 is 7054 Daltons), although a higher dose
of the PEP-1 KI polypeptide can be required if all of the plasma
kallikrein (500 nM) is activated and/or if this KI polypeptide is
poorly distributed in a patient.
[0061] Furthermore, the KI polypeptides can be non-naturally
occurring, and they can be produced synthetically or recombinantly,
as noted above, thereby avoiding potential contamination of
transmissible diseases that can arise during isolation of a protein
from a natural animal source, such as in the case of aprotinin,
which is isolated from bovine lung. Increasingly important to
administrative and public acceptance of a treatment or
pharmaceutical composition comprising a polypeptide is the
avoidance of possible contamination with and transmission to human
patients of various pathological agents. Of particular interest for
the safety of proteins isolated from a bovine tissue is the
elimination of the possible risk of exposure to viral mediated
diseases, bacterial mediated diseases, and, especially,
transmissible bovine spongiform encephalopathies.
[0062] As variants of the Kunitz domain 1 of the human LACI
protein, fewer side effects are expected from administering the KI
polypeptides to patients than for aprotinin, which is a bovine
protein that is documented to cause anaphylactic and anaphylactoid
responses in patients, especially in repeat administrations, such
as second time CABG procedures. Additionally, the highly specific
binding of the KI polypeptides described herein to kallikrein will
effectively limit or eliminate the thrombotic tendencies observed
with aprotinin, and reduce the problems observed with graft patency
following CABG procedures.
[0063] The invention will be further described with reference to
the following non-limiting examples. The teachings of all the
patents, patent applications and all other publications and
websites cited herein are incorporated by reference in their
entirety.
EXEMPLIFICATION
Example 1
A Representative KI Polypeptide
[0064] A non-naturally occurring, KI polypeptide useful in the
compositions and methods of the invention was identified as a
kallikrein binding polypeptide displayed on a recombinant phage
from a phage display library. PEP-1 has the following amino acid
sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly
Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln
Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu
Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2). The
molecular weight of PEP-1 is 7,054 Daltons.
[0065] The nucleotide sequence (SEQ ID NO:3) encoding the PEP-1
amino acid sequence (SEQ ID NO:2), was derived from a peptide that
was isolated and sequenced by standard methods determined from the
recombinant phage DNA. PEP-1 was produced in amounts useful for
further characterization as a recombinant protein in
His4.sup.-phenotype host cells of yeast strain Pichia pastoris.
Example 2
Construction of a Recombinant Plasmid to Express KI
Polypeptides
[0066] The initial plasmid, pHIL-D2, is ampicillin resistant and
contains a wild-type allele of His4 from P. pastoris. The final DNA
sequence comprising the coding sequence for the mat.alpha.
Prepro-PEP-1 fusion protein in the recombinant expression plasmid
pPIC-K503 is shown in FIG. 2. The DNA sequence of pHIL-D2 was
modified to produce pPIC-K503, as follows:
[0067] 1. The BstBI site in the 3' AOX1 region of pHIL-D2, located
downstream of the His4 gene, was removed by partial restriction
digestion, fill-in, and ligation, altering the sequence from TTCGAA
(SEQ ID NO:23) to TTCGCGAA (SEQ ID NO:24). This modification was
made to facilitate and direct the cloning of the expression
cassette into the plasmid.
[0068] 2. The AatII site bearing the bla gene located downstream of
His4 was removed by restriction digestion, fill-in, and ligation
modifying the sequence from GACGTC (SEQ ID NO:25) to GACGTACGTC
(SEQ ID NO:26). This modification was made to facilitate the
cloning of expression cassettes having AatII sites into the
plasmid. The DNA encoding PEP-1 was synthesized based on the
nucleotide sequence from the original kallikrein-binding display
phage and consisted of 450 base pairs (bp). The final DNA sequence
of the insert in the pHIL-D2 plasmid is flanked by a 5' AOX1
sequence and a 3' AOX1 sequence (portions of which are shown in
FIG. 2) and encode a fusion protein comprising the mat.alpha.
prepro signal peptide of S. cerevisiae fused to the structural
coding sequence for the PEP-1 KI polypeptide. The signal peptide
was added to facilitate the secretion of PEP-1 from the yeast host
cells. The oligonucleotides to form the insert were synthesized and
obtained commercially (Genesis Labs, The Woodlands, Tex.), and the
insert was generated by polymerase chain reaction (PCR). The linked
synthetic DNA encoding the mat.alpha. prepro/PEP-1 fusion protein
was then incorporated by ligation into the modified pHIL-D2 plasmid
between the BstBI and EcoRI sites.
[0069] The ligation products were used to transform Escherichia
coli strain XL1 Blue. A PCR assay was used to screen E. coli
transformants for the desired plasmid construct. DNA from cell
extracts was amplified by PCR using primers containing the 5' AOX1
and 3' AOX1 sequences (see above and FIG. 2). PCR products of the
correct number of base pairs were sequenced. In addition,
approximately 20-50 bp on either side of the cloning sites were
sequenced, and the predicted sequence was obtained. The final DNA
sequence of the insert in the pHIL-D2 plasmid (to yield plasmid
pPIC-K503) is shown in FIG. 2 along with portions of flanking 5'
and 3' AOX1 sequences and corresponding amino acid sequence of the
fusion protein comprising the mat.alpha. prepro signal peptide of
S. cerevisiae fused to the structural coding sequence for the PEP-1
KI polypeptide. A transformant with the desired expression plasmid
construct, plasmid pPIC-K503, was selected for preparing yeast cell
lines for routine production of PEP-1.
Example 3
Manufacture of PEP-1 from Recombinant Yeast Cell Line
[0070] Spheroplasts of P. pastoris GS115 having the
His4.sup.-phenotype were transformed with the expression plasmid
pPIC-K503 (above) following linearization of the plasmid at the
SacI site and homologous recombination of the plasmid DNA into the
host 5' AOX1 locus. The phenotype of the production strain is
His4.sup.+. The entire plasmid was inserted into the 5' AOX1
genomic sequence of the yeast.
[0071] Isolates from the transformation were screened for growth in
the absence of exogenous histidine with methanol as the sole carbon
source. Greater than 95% of the transformants retained the
wild-type ability to grow with methanol as the sole carbon source,
thereby demonstrating that the plasmid had been inserted into the
host genome by homologous recombination rather than transplacement.
These transformants did not require exogenous histidine for growth,
thereby demonstrating that the plasmid had integrated into the host
genome. Selected colonies were cloned. Small culture expression
studies were performed to identify clones secreting the highest
levels of active PEP-1 into the culture medium. PEP-1 secretion
levels in clarified culture supernatant solutions were quantified
for PEP-1 levels by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and evaluated for kallikrein inhibition.
A yeast clone was selected for PEP-1 production based on its high
level of PEP-1 expression among cultures sampled.
[0072] Master and working cell banks of P. pastoris producing PEP-1
were prepared commercially (MDS Pharma Services, Bothell, Wash.). A
standard production of PEP-1 in yeast comprised three steps as
follows: (1) preparation of the seed culture, (2) fermentation, and
(3) recovery of the culture.
[0073] The seed culture step consisted of the inoculation of six
flasks (300 mL each) containing sterile inoculum broth (yeast
nitrogen base, potassium phosphate, and glycerol, pH=5) with the
contents of a single vial of a working cell bank of P. pastoris
producing PEP-1. Flasks were inoculated in an orbital shaker (300
rpm) for approximately 13 hours at 30.degree. C.+-.2.degree. C.
[0074] Fermentations were performed in a closed 100 liter Braun
fermenter filled with sterile broth. Each fermentation was
initiated with the transfer of the contents of the six seed culture
flasks to the fermenter. After approximately 24 hours, the glycerol
in the fermenter became exhausted and additional glycerol was added
for approximately 8 additional hours.
[0075] A mixed feed phase, which lasted approximately 83 hours, was
then initiated by the addition of a glycerol and methanol feed. At
the end of this time, the fermentation was terminated, and the
fermenter contents were diluted with purified water. The
purification and processing of PEP-1 consisted of five steps as
follows: (1) expanded bed chromatography, (2) cation exchange
chromatography, (3) hydrophobic interaction chromatography (HIC),
(4) ultrafiltration and diafiltration, and (5) final filtration and
packaging.
[0076] The initial purification step consisted of expanded bed
chromatography. The diluted fermenter culture was applied to the
equilibrated column packed with Streamline SP resin (Amersham
Pharmacia Streamline 200 chromatography column, Amersham Pharmacia,
Piscataway, N.J.). The column was then washed (50 mM acetic acid,
pH=3.0-3.5) in an up-flow mode to flush the yeast cells from the
expanded bed. The top adaptor was raised above the expanded bed
enhance washing. The flow was stopped and the bed was allowed to
settle. The adaptor was moved down so that it was slightly above
the settled bed. The direction of the flow was reversed. The
effluent was collected. Washing was continued in a downward mode
using 50 mM sodium acetate, pH 4.0. The effluent was collected.
PEP-1 was eluted from the column using 50 mM sodium acetate, pH
6.0. The eluate was collected in a 50 liter container. The eluate
was then filtered through a 0.22.mu. filter into a clean container
located in the purification site. Additional samples were collected
for the determination of PEP-1 concentration. A cation exchange
chromatography step was then performed using the filtered eluate
from the expanded bed column. PEP-1 was eluted from the column
using 15 mM trisodium citrate, pH 6.2.
[0077] Additional proteins were removed from the PEP-1 preparation
by hydrophobic interaction chromatography (HIC). Prior to HIC, the
eluate from the cation exchange column was diluted with ammonium
sulfate. The eluate was applied to the column, and the PEP-1 was
eluted using ammonium sulfate (0.572 M) in potassium phosphate (100
mM), pH 7.0. The eluate was collected in fractions based on A280
values. All fractions were collected into sterile, pre-weighed PETG
bottles.
[0078] Selected fractions were pooled into a clean container. The
pool was concentrated by ultrafiltration. The concentrated PEP-1
preparation was immediately diafiltered against ten volumes of PBS,
pH 7.0.
[0079] A final filtration step was performed prior to packaging in
order to minimize the bioburden in the bulk PEP-1. The bulk
solution was filtered through a 0.22.mu. filter and collected into
a sterile, pre-weighed PETG bottle. A sample was removed for lot
release testing. The remainder of the bulk was dispensed
aseptically into sterile PETG bottles and stored at -20.degree.
C.
Example 4
Kallikrein Inhibition Assay
[0080] A kinetic test was used to measure inhibitory activity of KI
polypeptides, such as PEP-1. The kinetic assay measures
fluorescence following kallikrein-mediated cleavage of a substrate,
prolylphenylalanylarginyl amino methyl coumarin. A known amount of
kallikrein was incubated with a serially diluted KI polypeptide
reference standard or serially diluted KI polypeptide test samples,
in a suitable reaction buffer on a microtiter plate. Each sample
was run in triplicate. The substrate solution was added, and the
plate read immediately using an excitation wavelength of 360 nm and
an emission wavelength of 460 nm. At least two each of the
reference standard and sample curves were required to have an
R-squared value of 0.95 to be considered valid.
[0081] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
54 1 58 PRT Artificial Sequence Polypeptide Inhibiting Kallikrein
VARIANT 1, 2, 3, 4, 6, 7, 8, 9, 20, 24, 25, 26, 27, 28, 29, 41, 42,
44, 46, 47, 48, 49, 50, 52, 53, 54, 56, 57, 58 Xaa = any amino acid
VARIANT 10 Xaa = Asp or Glu VARIANT 11 Xaa = Asp, Gly, Ser, Val,
Asn, Ile, Ala or Thr VARIANT 13 Xaa = Arg, His, Pro, Asn, Ser, Thr,
Ala, Gly, Lys or Gln VARIANT 15 Xaa = Arg, Lys, Ala, Ser, Gly, Met,
Asn or Gln VARIANT 16 Xaa = Ala, Gly, Ser, Asp or Asn VARIANT 17
Xaa = Ala, Asn, Ser, Ile, Gly, Val, Gln or Thr VARIANT 18 Xaa =
His, Leu, Gln or Ala VARIANT 19 Xaa = Pro, Gln, Leu, Asn or Ile
VARIANT 21 Xaa = Trp, Phe, Tyr, His or Ile VARIANT 22 Xaa = Tyr or
Phe VARIANT 23 Xaa = Tyr or Phe VARIANT 31 Xaa = Glu, Asp, Gln,
Asn, Ser, Ala, Val, Leu, Ile or Thr VARIANT 32 Xaa = Glu, Gln, Asp,
Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val VARIANT 34 Xaa = Thr, Ile,
Ser, Val, Ala, Asn, Gly or Leu VARIANT 35 Xaa = Tyr, Trp or Phe
VARIANT 39 Xaa = Glu, Gly, Ala, Ser or Asp VARIANT 40 Xaa = Gly or
Ala VARIANT 43 Xaa = Asn or Gly VARIANT 45 Xaa = Phe or Tyr 1 Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Cys Xaa Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
20 25 30 Phe Xaa Xaa Gly Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 35 40 45 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 50 55 2 60
PRT Artificial Sequence Isolated Binding Peptide 2 Glu Ala Met His
Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys 1 5 10 15 Arg Ala
Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys 20 25 30
Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu 35
40 45 Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 60 3
179 DNA Artificial Sequence Coding Sequence of Pep-1 3 gaggctatgc
actctttctg tgctttcaag gctgacgacg gtcgtgcaga gctgctcacc 60
caagatggtt cttcaacatc ttcacgcgtc aatgcgagga gttcatctac ggtggttgtg
120 agggtaacca aaacagattc gagtctctag aggagtgtaa gaagatgtgt
actagagac 179 4 58 PRT Artificial Sequence Isolated Binding Peptide
4 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala 1
5 10 15 Asn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu
Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe
Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55
5 58 PRT Artificial Sequence Isolated Binding Peptide 5 Met His Ser
Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Asn
His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25
30 Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu
35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 6 58 PRT
Artificial Sequence Isolated Binding Peptide 6 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Asn His Gln
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30 Phe
Thr Tyr Gly Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 7 58 PRT
Artificial Sequence Isolated Binding Peptide 7 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Ser Leu Pro
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 8 58 PRT
Artificial Sequence Isolated Binding Peptide 8 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Asn His Gln
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 9 58 PRT
Artificial Sequence Isolated Binding Peptide 9 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly His Cys Lys Gly 1 5 10 15 Ala His Leu
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 10 58 PRT
Artificial Sequence Isolated Binding Peptide 10 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly 1 5 10 15 Ala His Leu
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 11 58 PRT
Artificial Sequence Isolated Binding Peptide 11 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Gly 1 5 10 15 Ala His Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 12 58 PRT
Artificial Sequence Isolated Binding Peptide 12 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala 1 5 10 15 Ala His Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 13 58 PRT
Artificial Sequence Isolated Binding Peptide 13 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly 1 5 10 15 Ala His Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 14 58 PRT
Artificial Sequence Isolated Binding Peptide 14 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly 1 5 10 15 Ala Gln Pro
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 15 58 PRT
Artificial Sequence Isolated Binding Peptide 15 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Ser Cys Arg Ala 1 5 10 15 Ala His Leu
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 16 58 PRT
Artificial Sequence Isolated Binding Peptide 16 Met His Ser Phe Cys
Ala Phe Lys Ala Glu Gly Gly Ser Cys Arg Ala 1 5 10 15 Ala His Gln
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 17 58 PRT
Artificial Sequence Isolated Binding Peptide 17 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly 1 5 10 15 Ala His Leu
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 18 58 PRT
Artificial Sequence Isolated Binding Peptide 18 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly His Cys Arg Gly 1 5 10 15 Ala Leu Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 19 58 PRT
Artificial Sequence Isolated Binding Peptide 19 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly 1 5 10 15 Asn Leu Pro
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 20 58 PRT
Artificial Sequence Isolated Binding Peptide 20 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg Gly 1 5 10 15 Asn His Gln
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 21 58 PRT
Artificial Sequence Isolated Binding Peptide 21 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Ala 1 5 10 15 Ile Gln Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 22 58 PRT
Artificial Sequence Isolated Binding Peptide 22 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Arg Cys Arg Gly 1 5 10 15 Ala His Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 23 6 DNA
Artificial Sequence Modified Cloning Site 23 ttcgaa 6 24 8 DNA
Artificial Sequence Modified Cloning Site 24 ttcgcgaa 8 25 6 DNA
Artificial Sequence Modified Cloning Site 25 gacgtc 6 26 10 DNA
Artificial Sequence Modified Cloning Site 26 gacgtacgtc 10 27 548
DNA Artificial Sequence Nucleotide Sequence of Fusion Protein 27
cgacttttaa cgacaacttg agaagatcaa aaaacaacta attattcgaa acgatgagat
60 tcccatctat cttcactgct gttttgttcg ctgcttcctc tgctttggct
gctccagtta 120 acaccactac tgaagacgag actgctcaaa ttcctgctga
ggctgtcatc ggttactctg 180 acttggaagg tgacttcgac gtcgctgttt
tgccattctc taactctact aacaacggtt 240 tgttgttcat caacactacc
atcgcttcta tcgctgctaa ggaggaaggt gtttccctcg 300 agaagagaga
ggctatgcac tctttctgtg ctttcaaggc tgacgacggt ccgtgcagag 360
ctgctcaccc aagatggttc ttcaacatct tcacgcgtca atgcgaggag ttcatctacg
420 gtggttgtga gggtaaccaa aacagattcg agtctctaga ggagtgtaag
aagatgtgta 480 ctagagacta gtaagaattc gccttagaca tgactgttcc
tcagttcaag ttgggcactt 540 acgagaag 548 28 145 PRT Artificial
Sequence Fusion Protein 28 Met Arg Phe Pro Ser Ile Phe Thr Ala Val
Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr
Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val
Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val
Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile
Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala 85
90 95 Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn
Ile 100 105 110 Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys
Glu Gly Asn 115 120 125 Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys
Lys Met Cys Thr Arg 130 135 140 Asp 145 29 58 PRT Artificial
Sequence BPTI Sequence 29 Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr
Thr Gly Pro Cys Lys Ala 1 5 10 15 Arg Ile Ile Arg Tyr Phe Tyr Asn
Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30 Phe Val Tyr Gly Gly Cys
Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45 Glu Asp Cys Met
Arg Thr Cys Gly Gly Ala 50 55 30 58 PRT Artificial Sequence ITI-D1
Sequence 30 Lys Glu Asp Ser Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys
Met Gly 1 5 10 15 Met Thr Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met
Ala Cys Glu Thr 20 25 30 Phe Gln Tyr Gly Gly Cys Met Gly Asn Gly
Asn Asn Phe Val Thr Glu 35 40 45 Lys Glu Cys Leu Gln Thr Cys Arg
Thr Val 50 55 31 58 PRT Artificial Sequence ITI-D2 Sequence 31 Thr
Val Ala Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Arg Ala 1 5 10
15 Phe Ile Gln Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu
20 25 30 Phe Pro Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr
Ser Glu 35 40 45 Lys Glu Cys Arg Glu Tyr Cys Gly Val Pro 50 55 32
58 PRT Artificial Sequence LACI-D1 Sequence 32 Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala 1 5 10 15 Ile Met Lys
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40
45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 33 58 PRT
Artificial Sequence LACI-D2 Sequence 33 Lys Pro Asp Phe Cys Phe Leu
Glu Glu Asp Pro Gly Ile Cys Arg Gly 1 5 10 15 Tyr Ile Thr Arg Tyr
Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg 20 25 30 Phe Lys Tyr
Gly Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu 35 40 45 Glu
Glu Cys Lys Asn Ile Cys Glu Asp Gly 50 55 34 58 PRT Artificial
Sequence LACI-D3 Sequence 34 Gly Pro Ser Trp Cys Leu Thr Pro Ala
Asp Arg Gly Leu Cys Arg Ala 1 5 10 15 Asn Glu Asn Arg Phe Tyr Tyr
Asn Ser Val Ile Gly Lys Cys Arg Pro 20 25 30 Phe Lys Tyr Ser Gly
Cys Gly Gly Asn Glu Asn Asn Phe Thr Ser Lys 35 40 45 Gln Glu Cys
Leu Arg Ala Cys Lys Lys Gly 50 55 35 58 PRT Artificial Sequence HKI
B9 Sequence 35 Leu Pro Asn Val Cys Ala Phe Pro Met Glu Lys Gly Pro
Cys Gln Thr 1 5 10 15 Tyr Met Thr Arg Trp Phe Phe Asn Phe Glu Thr
Gly Glu Cys Glu Leu 20 25 30 Phe Ala Tyr Gly Gly Cys Gly Gly Asn
Ser Asn Asn Phe Leu Arg Lys 35 40 45 Glu Lys Cys Glu Lys Phe Cys
Lys Phe Thr 50 55 36 58 PRT Artificial Sequence C alpha 3 Sequence
36 Glu Thr Asp Ile Cys Lys Leu Pro Lys Asp Glu Gly Thr Cys Arg Asp
1 5 10 15 Phe Ile Leu Lys Trp Tyr Tyr Asp Pro Asn Thr Lys Ser Cys
Ala Arg 20 25 30 Phe Trp Tyr Gly Gly Cys Gly Gly Asn Glu Asn Lys
Phe Gly Ser Gln 35 40 45 Lys Glu Cys Glu Lys Val Cys Ala Pro Val 50
55 37 58 PRT Artificial Sequence TFPI-2 D1 Sequence 37 Asn Ala Glu
Ile Cys Leu Leu Pro Leu Asp Tyr Gly Pro Cys Arg Ala 1 5 10 15 Leu
Leu Leu Arg Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln 20 25
30 Phe Leu Tyr Gly Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr Thr Trp
35 40 45 Glu Ala Cys Asp Asp Ala Cys Trp Arg Ile 50 55 38 61 PRT
Artificial Sequence TFPI-2 D2 Sequence 38 Val Pro
Lys Val Cys Arg Leu Gln Val Ser Val Asp Asp Gln Cys Glu 1 5 10 15
Gly Ser Thr Glu Lys Tyr Phe Phe Asn Leu Ser Ser Met Thr Cys Glu 20
25 30 Lys Phe Phe Ser Gly Gly Cys His Arg Asn Arg Ile Glu Asn Arg
Phe 35 40 45 Pro Asp Glu Ala Thr Cys Met Gly Phe Cys Ala Pro Lys 50
55 60 39 58 PRT Artificial Sequence TFPI-2 D3 Sequence 39 Ile Pro
Ser Phe Cys Tyr Ser Pro Lys Asp Glu Gly Leu Cys Ser Ala 1 5 10 15
Asn Val Thr Arg Tyr Tyr Phe Asn Pro Arg Tyr Arg Thr Cys Asp Ala 20
25 30 Phe Thr Tyr Thr Gly Cys Gly Gly Asn Asp Asn Asn Phe Val Ser
Arg 35 40 45 Glu Asp Cys Lys Arg Ala Cys Ala Lys Ala 50 55 40 59
PRT Artificial Sequence APP-I Sequence 40 Arg Asn Arg Glu Val Cys
Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg 1 5 10 15 Ala Met Ile Ser
Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala 20 25 30 Pro Phe
Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr 35 40 45
Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala 50 55 41 58 PRT
Artificial Sequence EpiNE7 Sequence 41 Arg Pro Asp Phe Cys Leu Glu
Pro Pro Tyr Thr Gly Pro Cys Val Ala 1 5 10 15 Met Phe Pro Arg Tyr
Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30 Phe Val Tyr
Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Lys Ser Ala 35 40 45 Glu
Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 42 58 PRT Artificial
Sequence BITI-E7-141 Sequence 42 Arg Pro Asp Phe Cys Gln Leu Gly
Tyr Ser Ala Gly Pro Cys Val Ala 1 5 10 15 Met Phe Pro Arg Tyr Phe
Tyr Asn Gly Thr Ser Met Ala Cys Gln Thr 20 25 30 Phe Val Tyr Gly
Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr Glu 35 40 45 Lys Asp
Cys Leu Gln Thr Cys Arg Gly Ala 50 55 43 58 PRT Artificial Sequence
MUTT26A Sequence 43 Arg Pro Asp Phe Cys Gln Leu Gly Tyr Ser Ala Gly
Pro Cys Val Ala 1 5 10 15 Met Phe Pro Arg Tyr Phe Tyr Asn Gly Ala
Ser Met Ala Cys Gln Thr 20 25 30 Phe Val Tyr Gly Gly Cys Met Gly
Asn Gly Asn Asn Phe Val Thr Glu 35 40 45 Lys Asp Cys Leu Gln Thr
Cys Arg Gly Ala 50 55 44 58 PRT Artificial Sequence MUTQE Sequence
44 Arg Pro Asp Phe Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Val Ala
1 5 10 15 Met Phe Pro Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys
Glu Thr 20 25 30 Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn
Phe Val Thr Glu 35 40 45 Lys Asp Cys Leu Gln Thr Cys Arg Gly Ala 50
55 45 58 PRT Artificial Sequence MUT1619 Sequence 45 Arg Pro Asp
Phe Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Val Gly 1 5 10 15 Met
Phe Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys Gln Thr 20 25
30 Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr Glu
35 40 45 Lys Asp Cys Leu Gln Thr Cys Arg Gly Ala 50 55 46 62 PRT
Artificial Sequence EPI-HNE-1 Sequence 46 Glu Ala Glu Ala Arg Pro
Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly 1 5 10 15 Pro Cys Ile Ala
Phe Phe Pro Arg Tyr Phe Tyr Asn Ala Lys Ala Gly 20 25 30 Leu Cys
Gln Thr Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn 35 40 45
Phe Lys Ser Ala Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 60 47
56 PRT Artificial Sequence EPI-HNE-2 Sequence 47 Ala Ala Cys Asn
Leu Pro Ile Val Arg Gly Pro Cys Ile Ala Phe Phe 1 5 10 15 Pro Arg
Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro 20 25 30
Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu 35
40 45 Cys Arg Glu Tyr Cys Gly Val Pro 50 55 48 56 PRT Artificial
Sequence EPI-HNE-3 Sequence 48 Ala Ala Cys Asn Leu Pro Ile Val Arg
Gly Pro Cys Ile Ala Phe Phe 1 5 10 15 Pro Arg Trp Ala Phe Asp Ala
Val Lys Gly Lys Cys Val Leu Phe Pro 20 25 30 Tyr Gly Gly Cys Gln
Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu 35 40 45 Cys Arg Glu
Tyr Cys Gly Val Pro 50 55 49 56 PRT Artificial Sequence EPI-HNE-4
Sequence 49 Glu Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Ile Ala
Phe Phe 1 5 10 15 Pro Arg Trp Ala Phe Asp Ala Val Lys Gly Lys Cys
Val Leu Phe Pro 20 25 30 Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys
Phe Tyr Ser Glu Lys Glu 35 40 45 Cys Arg Glu Tyr Cys Gly Val Pro 50
55 50 60 PRT Artificial Sequence DPI14 KR Sequence 50 Glu Ala Val
Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Ile
Ala Phe Phe Pro Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25
30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp
35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala 50 55 60
51 60 PRT Artificial Sequence DPI24 KR Sequence 51 Glu Ala Asn Ala
Glu Ile Cys Leu Leu Pro Leu Asp Tyr Gly Pro Cys 1 5 10 15 Ile Ala
Phe Phe Pro Arg Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys 20 25 30
Arg Gln Phe Leu Tyr Gly Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr 35
40 45 Thr Trp Glu Ala Cys Asp Asp Ala Cys Trp Arg Ile 50 55 60 52
60 PRT Artificial Sequence DPI68 KR Sequence 52 Glu Ala Lys Pro Asp
Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys 1 5 10 15 Ile Gly Phe
Phe Pro Arg Tyr Phe Tyr Asn Asn Gln Ala Lys Gln Cys 20 25 30 Glu
Arg Phe Val Tyr Gly Gly Cys Leu Gly Asn Met Asn Asn Phe Glu 35 40
45 Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu Asp Gly 50 55 60 53 60
PRT Artificial Sequence DPI84 KR Sequence 53 Glu Ala Glu Thr Asp
Ile Cys Lys Leu Pro Lys Asp Glu Gly Thr Cys 1 5 10 15 Ile Ala Phe
Phe Pro Arg Trp Tyr Tyr Asp Pro Asn Thr Lys Ser Cys 20 25 30 Ala
Arg Phe Val Tyr Gly Gly Cys Gly Gly Asn Glu Asn Lys Phe Gly 35 40
45 Ser Gln Lys Glu Cys Glu Lys Val Cys Ala Pro Val 50 55 60 54 58
PRT Artificial Sequence VARIANT VARIANT 10 Xaa = Asp or Glu VARIANT
11 Xaa = Asp, Gly, Ser, Val, Asn, Ile, Ala or Thr VARIANT 13 Xaa =
Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys or Gln VARIANT 15 Xaa =
Arg, Ala, Ser, Gly, Met, Asn or Gln VARIANT 16 Xaa = Ala, Gly, Ser,
Asp or Asn VARIANT 17 Xaa = Ala, Asn, Ser, Ile, Gly, Val, Gln or
Thr VARIANT 18 Xaa = His, Leu, Gln or Ala VARIANT 19 Xaa = Pro,
Gln, Leu, Asn or Ile VARIANT 21 Xaa = Trp, Phe, Tyr, His or Ile
VARIANT 31 Xaa = Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr
VARIANT 32 Xaa = Glu, Gln, Asp, Asn, Pro, Thr, Leu, Ser, Ala, Gly
or Val VARIANT 34 Xaa = Thr, Ile, Ser, Val, Ala, Asn, Gly or Leu
VARIANT 35 Xaa = Tyr, Trp or Phe VARIANT 39 Xaa = Glu, Gly, Ala,
Ser or Asp 54 Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa Gly Xaa
Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Arg Xaa Phe Phe Asn Ile Phe Thr
Arg Gln Cys Xaa Xaa 20 25 30 Phe Xaa Xaa Gly Gly Cys Xaa Gly Asn
Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp 50 55
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